One important function of intracellular proiein degradation is to selectively destroy proteins with abnormal conformations, whose accumulation can be toxic. Molecular chaperones, in addition to catalyzing protein folding and translocation, play several essential functions in this degradative process, which we hope to understand. Most protein breakdown in mammalian cells is catalyzed by the 26S proteasome, whose 19S regulatory particle contains six ATPases that function as molecular chaperones. To elucidate their roles in protein degradation, we are studying the simpler homologous ATPase complex, PAN, which supports proteolysis by 20S proteasomes in archaebacteria. We hope to clarify its mode of substrate recognition and how it catalyzes the unfolding of globular proteins and their translocation into the 20S proteasome. In eukaryotic cells, the chaperones hsp70 and hsp40 are required for the degradation of abnormal proteins by the ubiquitin-proteasome pathway, and recently a Ub ligase, CHIP, was discovered that functions with Hsp70 in the ubiquitination of unfolded proteins. We hope to define the precise roles of chaperones in ubiquitination by CHIP and to learn how additional factors, in particular, the ubiquitin-binding protein, S5a, may function to facilitate the degradation of the Ub-conjugated proteins by 26S proteasomes. In E. coli, the rapid degradation of certain polypeptides requires the chaperones, GroEL and GroES, which facilitate the unfolding of domains that resist degradation by ATP-dependent proteases. We shall test whether in eukaryotes there is a similar collaboration between the related chaperones (CCT/TriC) and proteasomes in catalyzing the complete degradation of "hard-to-unfold" domains in proteins. In harsh conditions which favor protein denaturation (e.g., heat-shock or oxidative stress), microorganisms produce hsps and large amounts of the "chemical chaperone," trehalose, which enhances cellular resistance to these stresses. We recently discovered that trehalose is also produced in high amounts upon shift to low temperatures (0-10 degrees C) in yeast, and its accumulation protects cells against freezing. We hope to learn how this disaccharide protects cell proteins against cold-induced denaturation, whether trehalose influences protein degradation, and whether higher eukaryotes show similar adaptations to near-freezing temperatures.